The hydration state in living cells is believed to be associated with various cellular activities. Nevertheless, in vivo characterization of intracellular hydration state under physiological condition has not been well documented to date. In this study, the hydration state of an intact HeLa cell monolayer was investigated by terahertz time-domain attenuated total reflection spectroscopy. Combined with the extended theory of Onsager, we found 23.8 ± 7.4% of HeLa intracellular water was hydrated to biomolecules (corresponding to 1.25 g H[2]O/g solute); exhibiting slower relaxation dynamics than bulk water.